Structure PA0 Function PA0 Anti-CD4 antibodies PA0 Effects of Anti-CD4 Antibodies: PA0 On antibody response PA0 On cell-mediated immunity PA0 Induction of tolerance PA0 Anti-CD4 antibodies as a therapy in autoimmune diseases PA0 Animal models PA0 Murine Diabetes PA0 Murine lupus erythematosus PA0 Other Experimental Diseases PA0 Anti-CD4 antibodies in clinical studies PA0 In Rheumatoid Arthritis PA0 In transplantation PA0 CD4 as a viral receptor PA0 Anti-CD4 monoclonal antibodies as diagnostic agents PA0 Oligonucleotide ligands that bind CD4 PA0 SELEX
Mature CD4 is a 55 kDa monomeric glycoprotein found primarily on a subset of T cells. Other cells that express CD4 include monocytes, macrophages and Langerhans' cells. The molecule consists of an extracellular region (370 aa), a hydrophobic transmembrane domain (26 aa) and a highly charged cytoplasmic domain (38 aa). The cytoplasmic domain is strongly conserved across the species of mammals. The extracellular and transmembrane region show about 55% homology between mouse and human (Maddon, et al., 1987; Littman, 1987). The extracellular region consists of four tandem domains (V1-V4) with sequence and predicted structural homology to immunoglobulin variable-joining (VJ-like) domains (Maddon, et al., 1985), and hence the protein has been classified as a member of the immunoglobulin gene family. The crystal structure of soluble CD4 (the extracellular region) shows a rod-like structure having a length of 125 .ANG. with a diameter of 25-30 .ANG. (Kwong, et al., 1990; Wang et al., 1990).
CD4 functions as an accessory molecule involved in the interaction between CD4.sup.+ T cells and antigen presenting cells (APC) that express MHC class II proteins. Thus CD4 functions as an adhesion molecule increasing the probability of aggregate formation between T cells and accessory cells (Doyle & Strominger, 1987). In addition to its role in cell adhesion, CD4 also acts as a coreceptor for the TCR/CD3 complex contributing to its signal transduction function. Several studies have shown that CD4 and CD3 undergo comodulation (Saizawa et al., 1987; Anderson et al., 1988) as well as coclustering (Kupfer et al., 1987) upon T cell activation. A model advanced that is consistent with these results is that there is a formation of microclusters within the cell membrane consisting of CD4 and TCR/CD3. According to this model, bispecific antibody constructs will allow the formation of microclusters and thereby activation of T cells. This was experimentally demonstrated by using bispecific antibody constructs capable of crosslinking CD3 and CD4 to activate T cells (Emmrich et al., 1986, 1987).
At least 25 different epitopes on CD4 have been postulated, and therefore the effect of any one antibody that binds to a specific epitope will be different from the effect of a second antibody targeted to a different epitope. Consequently, the effect of different monoclonal antibodies binding to CD4 differs with regard to mobilization of intracellular calcium when crosslinked to CD3, or to inhibition of anti-CD3 mediated stimulation. Anti-CD4 antibodies are able to block an antigen-stimulated T cell activation by: (1) inhibiting adhesion to accessory cells; (2) preventing microcluster formation between CD4 and TCR/CD3; and (3) exerting a negative signal. Anti-CD4 antibodies have been found to be most useful in inhibiting the initiation of T cell activation in resting T cells rather than the inhibition of activated T cell functions. Current understanding of the function of anti-CD4 monoclonal antibodies comes mainly from studies carried out with murine anti-CD4 monoclonal antibodies.
On lymphocytes
Most monoclonal antibodies induce severe CD4-selective lymphocyte depletion in blood, spleen and lymph but not the thymus after single antibody injection. The depletion persists for about 10 days and starts recovering and reaching completion after 4-6 weeks.
Monoclonal antibodies to CD4 inhibit primary and secondary antibody responses of IgM and IgG classes. This immunosuppressive effect is effective if the antigen is injected after anti-CD4 antibody treatment. The antigen injection can be delayed for up to 15 days after the antibody treatment (Goronzy et al., 1986).
Anti-CD4 monoclonal antibodies suppress delayed-type hypersensitivity reactions in normal as well as in C5-deficient mice, suggesting that complement is not a requirement (Kelley et al., 1987). Anti-CD4 monoclonal antibodies also inhibit the appearance of CD8.sup.+ cytotoxic T cells against allogenic cells and virus infected cells (Woodcock et al., 1986; Weyand et al., 1989). Prolongation of renal allograft survival in monkeys using murine and human CD4 monoclonal antibodies has been described (Jonker et al, 1985, 1987; Cosimi et al., 1991).
A unique property of certain anti-CD4 antibodies is their ability to induce immunological tolerance to both soluble and cellular antigens. However, this capacity is not a general feature of all anti-CD4 antibodies. Non-depleting anti-CD4 antibodies have induced tolerance in mice against human and rat immunoglobulins and allergenic bone marrow and skin grafts (Qin et al., 1990). Maintenance of the tolerance state to soluble antigens requires repeated injection, a condition which is not required for skin allografts that are not cleared like soluble antigens. The necessity of continuous antigen exposure probably indicates the involvement of T cells recently emerged from the thymus, since adult thymectomized mice remain tolerant to soluble antigens without reinforcement. The mechanism of anti-CD4 antibody induced tolerance is unclear.
In addition to the induction of tolerance, certain depleting anti-CD4 monoclonal antibodies do not sensitize, i.e., a rat monoclonal antibody does not elicit an antibody response in mice (Cobbold et al., 1984). The lack of sensitization is not directly due to immunosuppression, since it persists 42 days after injection of the antibody, after the time the mice have already recovered from immunosuppression (Benjamin & Waldman, 1986).
In general, anti-CD4 monoclonal antibodies may inhibit the onset or even stop the course of experimental autoimmune diseases.
The most studied spontaneous experimental model of insulin dependent diabetes mellitus (IDDM) is based on nonobese diabetic (NOD) mice. In this model, diabetes occurs in females at 4-6 months of age and is preceded by a long phase of clinically silent insulitis (T cell infiltration of the islets). The infusion of purified T cells derived from a diabetic mouse spleen to syngeneic recipients transfers the disease. The diabetes pathogenesis involves both CD4 and CD8 cells, since in vitro elimination of these cells at the time of transfer eliminates the disease transfer (Bendelec et al., 1987). A large proportion of diabetogenic T cell clones (capable of transferring the disease) expresses the CD4 phenotype (Haskins et al., 1989).
Certain Anti-CD4 antibodies prevent the onset of IDDM when the treatment is initiated at 3 months, provided that the injections are repeated weekly (Charlton & Mandel, 1988; Hayward et al., 1988). However, in the weeks following the cessation of therapy, reinfiltration of islets may occur without diabetes (hyperglycemia). NOD mice treated before the onset of insulitis (about 2 weeks) do not develop insulitis and diabetes (Koike et al., 1987).
Weekly injections of anti-CD4 monoclonal antibodies at the age of 4 months (i.e., prior to the onset of overt autoimmune disease) to NZB X NZW F1 mice reduced the titre of anti-DNA antibodies and prevented the onset of glomerulonephritis and renal failure (Wofsy & Seaman, 1985) with a clear prolonged survival time. More importantly, anti-CD4 monoclonal antibody therapy is still efficacious when started after the time of disease onset when high anti-DNA antibody titres and proteinuria are observed (Wofsy & Seaman, 1987). This result is directly relevant to therapy in humans.
Waldor et al., 1985, demonstrated that anti-CD4 monoclonal antibodies could prevent the development of experimental allergic encephalomyelitis (EAE) in the mouse and the rat.
Treatment with anti-CD4 monoclonal antibodies in experimental allergic myasthenia gravis has been shown to inhibit the disease (Christadoss & Dauphinee, 1986). The incidence of collagen type II induced arthritis in DBA/1-mice was also significantly reduced by the administration of anti-CD4 monoclonal antibodies (Hom et al., 1988).
The prevention and successful treatment of various autoimmune diseases in experimental models by anti-CD4 antibodies have encouraged the use of such reagents in clinical trials.
The first clinical trial including anti-CD4 monoclonal antibodies was done in a group of patients with rheumatoid arthritis (Herzog et al., 1987). After 7 consecutive days of treatment (10 mg of Antibody/day), 37 out of 46 patients showed improvement with respect to the number of swollen joints, pain, and Ritche's articular index. However, the level of rheumatoid factor and immune complex treatment did not change during or after the treatment. With a different anti-CD4 antibody, similar clinical efficacy as well as a significant decrease in total immunoglobulin levels, decreased levels of the rheumatoid factor and decreased levels of erythrocyte sedimentation rate were observed.
In these clinical trials, clinical responses were observed immediately after the treatment and lasted for more than 3 months. Even though these antibodies were murine in origin, they were well tolerated with low side effects and no noticeable immune suppression. Unlike in animals, no profound immunosuppressive effect has been seen in humans upon exposure to anti-CD4 antibodies (Dhiver et al., 1989). More than 50% of the patients who received mouse anti-CD4 antibodies developed low titre human anti-mouse antibody (HAMA) response. Partially humanized antibodies have also been tried in clinical trials with rheumatoid arthritis patients, and have been shown to be effective (van der Lubbe et al., 1991).
The pivotal role played by CD4.sup.+ T cells in allograft rejection was demonstrated in a mouse model (Cobbold et al., 1984; Madsen et al., 1987; Shizuru et al., 1990), and therefore represents a reasonable target for monoclonal antibody-based immunotherapy. The pretransplant injection of a murine anti-CD4 monoclonal antibody to rats selectively depleted 80-90% of peripheral CD4.sup.+ T cells and induced donor-specific tolerance of cardiac allografts (Shizuru et al., 1990). However, in nonhuman primates the clearance of peripheral CD4.sup.+ T cells was minimal as a result of delayed allograft rejection (Cosimi et al., 1990). The discrepancy between the rat and primate model may stem from the generation of an anti-murine response in primates due to the relatively distant phylogenic relationship. To overcome the anti-murine response in primates, murine antibodies have been humanized and such antibodies have been shown to be effective in the Cynomolgus renal allograft model (Powelson et al., 1994).
Initial attempts in using anti-CD4 antibodies in clinical trials were made in patients undergoing kidney transplantation (Morel et al., 1990) and the results are encouraging for further use of anti-CD4 antibodies in the management of rejection crises (Sabilinski et al., 1991).
Human immunodeficiency virus (HIV) utilizes the CD4 molecule as a mode of entry to infect T-helper cells. It has been shown that the envelope glycoprotein of HIV, gp120 , binds with high affinity to the V1 domain of CD4 (Dalgeish et al. 1984; Maddon et al. 1986). Extensive genetic and biochemical analyses have shown that gp120 binds to a 12 amino acid region encompassing the predicted c'c" loop which is analogous to the CDR2 (complementary-determining region) loop of an immunoglobulin. HIV infection is associated with the progressive decline of CD4.sup.+ T-cell subset (DeWolf et al. 1988). The decline of CD4.sup.+ T-helper cells could be due to the result of either virus-induced cell killing or CD4 down modulation in HIV-infected cells by viral gene products. As the absolute CD4.sup.+ cell count declines below 400/mm.sup.3, most patients show symptoms of AIDS. Thus, the absolute CD4 count is being used as a surrogate marker for the disease progress of HIV-infected individuals.
Anti-CD4 monoclonal antibodies are being used extensively to identify the T-helper subset in lymphocyte populations--especially in HIV-infected individuals--to follow disease progression and to obtain the decision point for initial anti-viral therapy (National Institute of Health, 1990). In addition, they have been useful in identifying T-cell activation (Maino et al. 1995). The preferred technique for using such antibodies has been multiparameter flow cytometry, where antibodies conjugated to different fluorophores are used.
Compounds having a polyanionic nature--such as sulfated polysaccharides (Weaver et al., 1990, 1992; Lederman et al. 1989), and dyes, like evans blue and tryphan blue (Balzarini et al. 1986)--have been shown to inhibit the binding of the HIV virus to T cells, and thereby the inhibition of virus-induced syntcytium formation. Oligonucleotides, being polyanionic, have also been shown to be inhibitory in viral entry (Matsakura et al. 1989 and references thereof). Although no complete and thorough study has been done to investigate the sequence specificity of oligonucleotides interfering with the gp120-CD4 interaction, the inhibitory effect has been shown to be length dependent (Stein et al. 1991). Oligonucleotides bearing phosphorothioate linkers in place of the regular phosphodiester linkage have identical charges, yet regular phosphodiester oligonucleotides do not inhibit syncytium formation. Compared to phosphodiesters, phosphorothioate oligonucleotides are nuclease resistant, a feature that is relevant to cellular assays. Phosphorothioate oligonucleotides, specifically a 28-mer consisting of cytosines (sdC28), bind both gp120 and CD4 with Kds in the .mu.M range (Stein et al., 1993). Phosphorothioate oligonucleotides bind to these two proteins as evidenced by experiments using oligonucleotides derivatized with an alkylating agent as a probe.
A method for the in vitro evolution of Nucleic Acid molecules with highly specific binding to Target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned; U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,475,096; U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also PCT/US91/04078), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a Nucleic Acid Ligand to any desired Target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of Nucleic Acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the Target under conditions favorable for binding, partitioning unbound Nucleic Acids from those Nucleic Acids which have bound specifically to Target molecules, dissociating the Nucleic Acid-Target complexes, amplifying the Nucleic Acids dissociated from the Nucleic Acid-Target complexes to yield a ligand-enriched mixture of Nucleic Acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity Nucleic Acid Ligands to the Target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," now abandoned, describes the use of SELEX in conjunction with gel electrophoresis to select Nucleic Acid molecules with specific structural characteristics, such as bent DNA (see, U.S. Pat. No. 5,707,796). U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands" now abandoned, describes a SELEX based method for selecting Nucleic Acid Ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a Target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," now abandoned, describes a method for identifying highly specific Nucleic Acid Ligands able to discriminate between closely related molecules, termed Counter-SELEX (See, U.S. Pat. No. 5,580,737). U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX," now abandoned, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a Target molecule (see, U.S. Pat. No. 5,567,588). U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled "Methods of Producing Nucleic Acid Ligands" now U.S. Pat. No. 5,496,938, describes methods for obtaining improved Nucleic Acid Ligands after SELEX has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar.8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX," now U.S. Pat. No. 6,705,337 describes methods for covalently linking a ligand to its Target.
The SELEX method encompasses the identification of high-affinity Nucleic Acid Ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified Nucleic Acid Ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," now abandoned, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines (see, U.S. Pat. No. 5,660,985). U.S. patent application Ser. No. 08/134,028, supra, describes highly specific Nucleic Acid Ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2' Modified Nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX", now U.S. Pat. No. 5,638,867 and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX," respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.